The isolation and identification of nucleic acids are important steps in many biochemical detection and clinical diagnostic tests. The separation of nucleic acids from the complex cellular compositions in which they are found is often a necessary initial step before detection or amplification can be undertaken. The presence of large amounts of cellular debris, such as proteins and carbohydrates, in the compositions often impedes the reactions and techniques used in molecular biology. The presence of exogenous agents frequently used for nucleic acid isolation can also inhibit nucleic acid amplification. Therefore, the current isolation and amplification procedures are undesirably time consuming, complicated, and inefficient. Thus, improved methods for the isolation and detection of nucleic acids, are desirable, for a broad variety of applications in medical diagnostics for microbial infections, detection of genetic variations, forensic science, tissue and blood typing, detection of environmental pathogens, and basic research, to name a few.
A range of methods are known for the isolation and purification of nucleic acids, but generally, these rely on a complex series of extraction and washing steps and are time consuming and laborious to perform. Classical methods for the isolation of nucleic acids from complex starting materials, such as blood, blood products, tissues, or other biological materials, involve lysis of the biological material, followed by isolation strategies such as solid phase extraction or phenol extraction followed with ethanol precipitation.
Paramagnetic bead technology has also been used for nucleic acid isolation. Most magnetic bead-based methods rely on lysing the sample followed by binding nucleic acids with magnetic beads and washing. Isolated nucleic acids obtained from these methods often contain agents, such as ethanol, which inhibit further amplification and detection.
Size-exclusion chromatography (SEC), also called gel-filtration or gel-permeation chromatography (GPC), uses porous particles stacked within a column to separate molecules of different sizes. It is generally used to separate biological molecules, and to determine molecular weights and molecular weight distributions of polymers. Molecules that are smaller than the pore size can enter the particles and therefore have a longer path and longer transit time than larger molecules that cannot enter the particles. Molecules larger than the pore size can not easily enter the pores, and elute together earlier in the chromatogram. Molecules that can enter the pores have an average residence time in the particles that depends on the molecular size and shape. Different molecules therefore have different total transit times through the column.
There is still a need for improved nucleic acid purification and detection methods which are quick, economical and simple to perform, which enable detectable yields to be obtained with minimal losses, whereby the nucleic acids obtained are ready for downstream amplification and analysis.